When an immune cell interacts with a target cell, and receptors bind their cognate ligands, a decision is made to react or not. Our long-range goal is to understand the basis of that decision. How does a cell monitor events at the cell surface? What gets counted, and how are multiple stimulatory and inhibitory signals integrated? How is that compared to a threshold value, and during development and education processes, how does a cell know what the threshold is? And once set, can these thresholds be manipulated? Each of these questions has major biomedical significance, from understanding the changes in reactivity that lead to autoimmunity, to inducing tolerance to a transplanted tissue. We have the ability to study how these processes work during a single immune interaction. Our model is histocompatibility in the basal chordate Botryllus schlosseri. Allorecognition in Botryllus is controlled by a single locus (called the fuhc) with the following rules: individuals that share one or both alleles are compatible; while those that share none are incompatible. Discrimination is based on the detection of a self-allele, and the specificity of this system is significant: there are ca. 1000 fuhc alleles world-wide, thus the effector system can pick out a self-allele from a sea of competing specificities. However, Botryllus does not have any recombination or somatic hypermutation machinery, and this specificity relies on germline-encoded receptors. This proposal is focused on understanding the biochemical mechanisms that underlie this innate allorecognition specificity. The unique properties of Botryllus allorecognition make it an ideal model for these studies: a single locus that determines outcome, the reaction occurs outside the body between epithelial cells on the tips of macroscopic blood vessel, called ampullae, and the outcome is determined by the integration of signaling pathways from only two receptors, one activating, and one inhibitory, both of which can be manipulated in vivo. In Aim 1, we will use a novel fluorescent labeling technique recently developed in our lab to isolate single ampullae cells and directly assess the basis of specificity. Our working hypothesis is that this is due to genotype-specific alternative splicing of a receptor called fester, and that will be directly tested here. Using this technique, we have also found that ampullae are bifunctional and can reversibly de-differentiate into vascular cells, which do not express allorecognition proteins, allowing us to characterize reversible changes in candidate protein expression/alternative splicing, which will reveal the basis of specificity. In Aim 2, we will assess extracellular ligand/receptor interactions in vivo and in vitro. We will test putative intracellular interactions between receptors and fuhc-encoded chaperones and scaffolding proteins that may play a role in creating receptor complexes and contribute to specificity. In Aim 3, we will characterize the signal transduction pathways used in Botryllus allorecognition, using a combination of FACS and proteomics. We have found that signal transduction molecules such as Zap-70, LCK, sph-1/2 and SHIP are expressed in ampullae. These genes have an early evolutionary origin, leading us to hypothesize that the conserved aspects of allorecognition are the mechanisms that integrate these well-characterized activating and inhibitory signal transduction pathways. However, integration with Aim 1 will also allow us take an unbiased view of putative signal transduction genes. Completion of the proposed aims will advance our understanding of the mechanisms that underlie education and tolerance in innate allorecognition systems. These intracellular processes monitor binding events at the cell surface, integrate activating and inhibitory inputs, and set and maintain the threshold for a response: the basis of specificity. We hypothesize that these are the conserved aspects of immunity, have an early evolutionary origin, and are responsible for the rapid evolutionary change characteristic of immunity- and Botryllus presents a unique and highly simplified model to study these processes. Understanding and manipulating threshold responses would be the building blocks of future clinical interventions, including inducing tolerance following transplantation, blocking autoimmune reactions, or overcoming the immunosuppressive strategies of tumors, areas of great importance for human health.